Translation termination is signaled by three stop codons: UAA, UAG, and UGA. This mechanism is highly conserved, although each stop codon has a different efficiency for terminating translation. UGA is considered to be a “leaky” stop codon with the highest intrinsic readthrough potential. UAA shows high fidelity and little intrinsic readthrough potential, whereas UAG has intermediate fidelity (see, e.g., Weiner and Weber, 1973, J. Mol. Biol. 80:837-855). Nonsense mutations create primary premature termination codons (PTCs) and result in either no formation of the target protein or truncated protein with impaired stability. Large numbers of genetic disorders are caused by nonsense mutations for which compound-induced readthrough of premature termination codons (PTCs) can be exploited as a potential treatment strategy.
Certain compounds influence the fidelity of stop codon recognition and induce readthrough of primary PTCs, which allows translation of some full-length protein. In many cases, the readthrough-induced protein is functional, even when it contains a wrongly incorporated amino acid (Keeling and Bedwell, 2005, Curr. Pharmacogenetics 3:259-269; Zingman et al, 2007, Clin. Pharmacol. Ther. 81:99-103).
It is estimated that 30% of human disease-causing alleles are nonsense mutations (Du et al, 2009, J. Exp. Med., 206 (10): 2285). Other types of mutations, such as frameshift and splicing mutations, lead to secondary PTCs; however, these are not therapeutic targets for readthrough compounds (RTCs). Considering that >1,800 distinct genetic disorders are caused by nonsense mutations, the readthrough of primary PTCs has treatment potential for large numbers of patients.
To date, most reported PTC-RTCs that are active in mammalian cells have belonged to the aminoglycoside antibiotics class (Keeling and Bedwell, 2005, Curr. Pharmacogenomics. 3:259-269; Zingman et al., 2007, Clin. Pharmacol. Ther., 81:99-103). Certain types of aminoglycosides can induce ribosomes to read through PTC mutations via insertion of a random amino acid by near-cognate transfer RNA. The therapeutic potential of aminoglycosides has been evaluated in the laboratory for different genetic models, such as cystic fibrosis (see, e.g., Du et al, 2002, J. Mol. Med. 80:595-604), muscular dystrophy (see, e.g., Loufrani et al, 2004, Arterioscler. 7hromb. Vase. Biol. 24:671-676), Hurler syndrome (Keeling et al, 2001, Hum. Mol. Genet. 10:291-299), cystinosis (Helip-Wooley et al, 2002, Mol. Genet. Metab. 75:128-133), spinal muscular atrophy (Sossi et al, 2001, Eur. J. Hum. Genet. 9:113-120), ataxia-telangiectasia (Lai et al., 2004, Proc. Natl. Acad. Sci. USA. 101:15676-15681), and type 1 Usher syndrome (Rebibo-Sabbah et al, 2007, Hum. Genet. 122:373-381). Clinical trials also indicate that aminoglycosides can induce some functional protein production; however, the therapeutic benefits remain uncertain (see, e.g., Politano et al., 2003, Acta Myol. 22: 15-21). Furthermore, the toxicity of most commercial aminoglycosides in mammals has greatly diminished their potential for successful readthrough therapy (Mingeot-Leclercq and Tulkens, 1999. Antimicrob. Agents Chemother. 43:1003-1012; Guan et al, 2000, Hum. Mol. Genet. 9:1787-1793). Therefore, efforts are underway to develop better aminoglycoside derivatives with reduced toxicity and enhanced activity (Nudelman et al, 2006, Bioorg. Med. Chem. Lett. 16:6310-6315; Rebibo-Sabbah et al., 2007, Hum. Genet. 122:373-381).
Recently, PTC Therapeutics (South Plainfield, N.J.) described a more efficient non-aminoglycoside RTC, PTC 124, which was developed synthetically by screening >800,000 chemicals and analogues using a luciferase-based high-throughput screening (HTS) assay (see, e.g., Welch et al, 2007, Nature. 447:87-91). A phase-I clinical study in cystic fibrosis confirmed that PTC124 is generally well tolerated and appears to have more efficient readthrough activity than aminoglycosides (Hirawat et al., 2007, J. Clin. Pharmacol. 47:430-444). Moreover, PTC124 does not induce ribosomal readthrough of normal stop codons. A phase-II clinical trial is underway (Kerem et al, 2008, Lancet. 372:719-727). However, a recent study indicates that the initial discovery of PTC 124 by HTS can have been biased by its direct effect on the FLuc (firefly luciferase) reporter used (Auld et al, 2009, Proc. Natl. Acad. Si. USA. 106:3585-3590), indicating the importance of a luciferase-independent HTS assay for future drug screening.
Recent findings have demonstrated that the specificity of the HTS utilized for the identification of the read-through compound may have been compromised by the ligand-induced stabilization of the reporter protein used for the screen (Auld, et al, (2009), Proc. Natl. Acad Sci. 106, 3585-3590). Although other interpretations have been offered (Peltz et al, P (2009), Proc. Natl. Acad Sci. USA, 106, E64), strong evidence have been presented in support of the post-translational activity of PTC 124 (Auld et al., 2010, Proc. Natl. Acad. Sci. USA. 107, 4878-4883; Thorne et al, 2010, Chem. Biol., 17, 646-657). Despite the off-target effects, PTC 124 has been shown to suppress nonsense mutations in different disease models and has reached clinical testing in patients (Sermet-Gaudelus et al., 2010, Am. J. Respir. Crit. Care Med. Vol. 182, No. 10, pp. 1262-1272; Welch et al, 2007, Nature. 447, 87-91). However, the indeterminate efficacy of Ataluren (PTC 124) in clinical trials for Duchenne boys supports the need of identifying new drugs that could be used to suppress nonsense mutations in the clinical scenario.
Recently, a sensitive and quantitative high-throughput screening method was developed by Du et al. (Du et al, 2009, J. Exp. Med. 206, 2285-2297) and used to screen low-molecular mass non-aminoglycoside compound libraries to identify potential drugs with PTC read-through activity. The screening protocol involved the use of a protein transcription/translation (PTT)-enzyme-linked immunosorbent assay (ELISA), using ataxia-telangiectasia (A-T) as a genetic disease model. This PTT-ELISA was driven by plasmid templates containing prototypic Premutations, patterned after specific disease-causing A-T (Du et al, 2009, J. Exp. Med. 206, 2285-2297). The screen of nearly 34,000 compounds led to the identification of compound 5 (provided in FIG. 1). This compound was shown to have biological activity in different lymphoblastoid cell lines derived from A-T patients containing each of the three types of nonsense mutations (TGA>TAA>TAG). Furthermore, compound 5 restored full-length dystrophin in mdx cells in culture (Du et al, 2009, J. Exp. Med 206, 2285-2297). Altogether, these data demonstrated that the compound has read-through activity on different types of proteins, in more than one species and cell lineage, and that activity is independent of the location of the premature stop codon within the transcript. Furthermore, compound 5 was shown to not read-through normal termination codons, thus confirming specificity for PTCs (Du et al, 2009, J. Exp. Med. 206, 2285-2297).
We have recently demonstrated that systemic administration of compound 5 restores functional levels of dystrophin expression in skeletal muscles of the mdx mice (Kayali et al., 2012, Hum. Mol. Genet., 21, 4007-4020):
Dystrophin protein was detected in all muscle groups analyzed, including diaphragm and heart, two of the muscles most difficult muscle to target by any of the therapeutic approaches currently being developed. Dystrophin was significantly higher than that achieved by PTC 124 and resulted in a significant increase in muscle strength over mice that did not receive the RTCs. The improvement in muscle function was paralleled by a decrease in creatine kinase (CK) levels a marker of muscle degeneration. These data demonstrate that compound 5 is a valid drug for the treatment of Duchenne muscular dystrophy (DMD) and suggest that many other disorders can benefit from its successful development into a drug.
However, one of the major limitations in further developing compound 5 for clinical applications is its low solubility.